Method for detecting species and biovars of Brucella

ABSTRACT

A method for detecting Brucella infection in an animal which is reliable, rapid, and able to identify species and biovars of Brucella. The detection method includes the amplification of the omp2 gene locus of Brucella and analysis of restriction digestion fragments specific to Brucella and to individual species and groups of biovars of Brucella.

FIELD OF THE INVENTION

This invention relates to a method for the diagnostic detection of thepathogenic bacterium Brucella. and more specifically to a method whichcan distinguish between species and biovars of Brucella.

BACKGROUND OF THE INVENTION

Brucella is a genus of pathogenic bacteria which cause acute or chronicillness in many animal species, including humans and cattle. Six speciesof Brucella and multiple biovars have been characterized by phenotypicmethods, although such methods are not always reliable. The six speciesand multiple biovars of Brucella may also be characterized by theirnatural host and a strain's geographical origin (See Table 1), however,a species may infect an animal other than its natural host, and a singlestrain may now be found in multiple geographic locations.

Early detection and characterization of the species or biovar of theinfecting Brucella organism would be of great value in medical andveterinary practice. Rapid and reliable detection of Brucella infectionis important to permit removal of infected cattle from a healthy herdand prevent the spread of the disease. Characterization of the speciesor biovar of Brucella would provide epidemiological data to determinethe source of the infection.

                  TABLE 1                                                         ______________________________________                                        SPECIES  BIOVAR    STRAIN     HOST   ORIGIN                                   ______________________________________                                        B. abortus                                                                             1         19                U.S.                                              1         2308       cattle U.S.                                              1         RB51       d.2308.sup.a                                                                         U.S.                                              1         45/20      d.45/0 England                                           2         ATCC 23449 cattle/                                                                              England                                           3         ATCC 23450 bison  Uganda                                            4         ATCC 23451 "      England                                           5         ATCC 23452 "      England                                           6         ATCC 23453 "      Africa                                            7         ATCC 23454 "                                                        9         ATCC 23455 "      England                                  B. melitensis                                                                          1         ATCC 23456 goat   U.S.                                     B. suis  1         ATCC 23444 pig    U.S.                                     B. neotomae        ATCC 23459 desert U.S.                                                                   wood rat                                        B. canis           ATCC 23365 dog    U.S.                                     B. ovis            ATCC 25840 sheep  Africa                                   ______________________________________                                         ATCC  American Type Culture Collection, Bethesda, Maryland.                   d.  derivative                                                           

Heretofore, standard serological tests used to detect Brucella haverequired several weeks time to complete and have not been able todistinguish between species of Brucella. The methods currently availableto identify species of infecting Brucella require the isolation ofbacteria on selective media followed by quantitative analysis ofphenotypic properties of the organism. Phenotypic characterization maybe based on such features as lipopolysaccharide antigens, phage typing,dye sensitivities, CO₂ requirements, H₂ S production, and metabolicproperties. Such methods are time consuming (requiring 1-4 weeks) andare unreliable. (see Alton, 1988, Techniques for the BrucellosisLaboratory; Moriera-Jacob, 1963, Nature 197:406; Shibata, 1962, Nat.Inst. Anim. Health Q. 2:10-14) Time delays in obtaining test results anduncertainty due to unreliable test results can result in great economiclosses. Suspect animals may require quarantine or may contaminatehealthy animals in the herd during the waiting period.

Identification of Brucella species using DNA probes has not previouslybeen possible, due to the high degree of inter-species DNA homology(approximately 90%).

There remains a great need for a rapid and accurate method for detectingthe presence of pathogenic Brucella organisms in a suspect animal. It isalso greatly desirable that such a detection method have the ability todistinguish between and identify the species and/or biovars of Brucella.

SUMMARY OF THE INVENTION

The method of the present invention solves the problems of the prior artmethods by providing a rapid, sensitive, and accurate diagnostic methodfor the detection of Brucella and, more specifically a diagnostic methodwhich is able to distinguish between species and biovars of Brucella.

It has now been found that the opm2 gene locus is conserved in allspecies of Brucella. Rapid detection of Brucella is achieved byidentification of the conserved omp2 gene locus.

It has also been found that genetic variation at the omp2 gene locus ofBrucella correlates with established species designations, and that thisgenetic variation may be used as a stable diagnostic marker forparticular species of Brucella. Differentiation between species andbiovars is based upon analysis of restriction fragment lengthpolymorphism in the omp2 gene locus of Brucella.

A preferred embodiment of the method of the present invention includesamplification of the omp2 gene locus. The amplified omp2 gene locus maythen be analyzed directly by electrophoretic separation, dot blot orSouthern Blot analysis to enable diagnosis of Brucella infection.Alternatively, restriction digestion of the amplified omp2 gene releasesfragments which may be analyzed, for example by gel electrophoresis, andthe restriction fragment pattern used to detect the presence of Brucellaand to identify the species or biovar of Brucella.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partial restriction map of the omp2 locus of the differentBrucella species and biovar groups.

FIG. 2 shows the top strand of the DNA sequence encoding the Brucellaabortus omp2 gene locus with solid lines depicting oligonucleotidesuseful in amplifying portions of the omp2 gene locus.

FIG. 3 is a partial restriction map of the omp2 locus of the Brucellaabortus.

FIG. 4 is a Southern Blot of Brucella genomic DNA digested with Bam HIand hybridized with a labeled Bam HI fragment containing the omp2 genelocus of B. abortus.

FIG. 5 is a Southern Blot of Brucella genomic DNA digested with Pst Iand hybridized with a labeled Bam HI fragment containing the omp2 genelocus of B. abortus.

FIG. 6 is an agarose gel stained with ethidium bromide showing thepresence of the amplified omp2 gene in Brucella infected versusnon-infected cattle.

FIG. 7 is a listing of oligonucleotides useful in the present invention.

DETAILED DESCRIPTION OF THE INVENTION

According to the method of the present invention, animal fluids ortissues may be tested for the presence of Brucella, and the species andbiovar of Brucella infecting the animal may be rapidly and accuratelydetected. Animal fluids and tissues including blood, urine, milk, semen,vaginal secretions, rectal secretions or other available tissues may becollected and used as the test sample, despite the presence of complex,non-Brucella DNA. The live bacteria in the sample are killed, forexample by heating to 68° C. for approximately 1 to 2 hours. The cellsof the sample are then lysed to release DNA, for example, by heating toapproximately 95° C. for approximately ten minutes or by repeatedfreezing and thawing of the cells. It may be desirable to immobilize thereleased DNA on a solid support in order to concentrate the DNA. Forexample, the DNA released by the lysed cells may be collected andconcentrated in an agarose gel, or on a nitrocellulose filter.

A desired gene sequence in the DNA released from the lysed cells is thenamplified, preferably through 30 to 50 cycles, by means of standardliquid polymerase chain reaction (PCR) using commercially availablecyclers or manually in changing water baths. The PCR method is known inthe art, and is described, for example, in Saiki et al, Science239:487-491, 1985, which is hereby incorporated by reference. Ingeneral, the PCR amplification method includes the hybridization of apair of oligonucleotide primers to a segment of DNA. The oligonucleotideprimers are designed to anneal to the DNA sequences flanking the targetgene sequence that is to be amplified, with one oligonucleotide upstreamand one downstream of the target sequence, on opposing DNA strands.During each amplification cycle, DNA strands are separated, for exampleby heating, priming oligonucleotides are annealed, for example bycooling the heated DNA in the presence of the oligonucleotides, and theprimers are extended using DNA polymerases and adding nucleotides to theend of each primer to make copies of the target DNA sequence. Thisprocess is repeated through approximately 30-50 amplification cycles,geometrically increasing the number of copies of the target genesequence.

Specific oligonucleotides are used to prime the amplification at theomp2 gene locus. As shown in FIG. 1, the omp2 gene locus includes theomp2a and omp2b genes as well as flanking and intervening genesequences. The DNA sequence of the B. abortus omp2 gene locus is shownin FIG. 2. Specific oligonucleotide pairs designed to hybridize tospecific gene sequences of the omp2 gene locus permit amplification of adesired gene sequence of the omp2 gene locus.

Examples of oligonucleotides useful in the present invention includethose listed in FIG. 7, and those shown in FIG. 2.

The amplified DNA may be analyzed directly by dot blot analysis using alabeled omp2 gene probe, by competitive hybridization analysis usingradiolabeled oligonucleotide probes, or by separating the amplified DNA,for example, using agarose gel electrophoresis and ethidium bromidestaining or Southern Blot analysis to detect the amplified genesequence. The presence of the amplified omp2 gene indicates the presenceof Brucella organisms in the test sample.

In a preferred embodiment, the amplified DNA may first be digested withspecific restriction enzymes to generate restriction fragmentscharacteristic of the omp2 gene locus prior to analysis by separationand staining or hybridization to specific omp2 gene probes. Properselection of the restriction enzyme may result in fragments displayingan electrophoretic pattern characteristic of the Brucella omp2 gene inall species of Brucella. Alternatively, the selection of restrictionenzymes may result in fragments displaying restriction fragment lengthpolymorphism (RFLP), for example, in the omp2a gene and flankingsequence of Brucella.

A preferred restriction enzyme which can be used to detect the omp2 genein all species of Brucella is Bam H1. Restriction digestion of genomicor amplified Brucella DNA using Bam HI releases a characteristic 6.5 kbfragment containing the omp2 gene.

Preferred restriction enzymes which can be used to identify theparticular species or biovar of the infecting Brucella organism includePstI and KpnI. Digestion of the amplified omp2 gene locus with PstIand/or KpnI results in restriction fragments displaying a uniqueelectrophoretic pattern in agarose gels for B.abortus, B. melitensis, B.canis, and B.ovis. The restriction fragment patterns for B. suis and B.neotomae, while distinct from the other 4 Brucella species, are notdistinguished from each other using these digestive enzymes. Biovars 1,2, and 4 of B.abortus may also be identified based upon the size of thePstI restriction fragments.

The pattern of restriction fragments may be visualized in theelectrophoretic gel by staining, for example, with ethidium bromide,which has a sensitivity in the range of 0.1-1.0 μg DNA. Alternate theamount of DNA is limited, i.e., 0.0-0.1 μg DNA, Southern Blot or dotblot analysis with omp2 DNA probes can be used.

EXAMPLES EXAMPLE 1 Conservation of the omp2 Gene Locus in Species andBiovars of Brucella

B.abortus smooth strains 19 and 2308 were obtained from Dr. Billy Deyoeat the National Animal Disease in Ames, Iowa. B.abortus biovars 1-7 and9, B.suis, B.canis. B.neotomae, B.melitensis, and B.ovis were obtainedfor the American Type Culture Collection, in Bethesda, Md. (See Table1). Strain identification was confirmed by standard biovar analysis (seeAlton, 1988). Brucella strains were cultivated on either Brucella agaror tryptic soy agar (Difco Laboratories, Detroit, Mich.). E. Coli cellswere grown as described in Ficht, 1988, Infect. Immunol. 56:2036-2046.

Brucella cells were grown on agar plates at 37° C. for approximately 48hours. Cells were washed off the plates in 5 ml of phenol/saline (0.1%w/v and 0.85% w/v, respectively). The cells were killed by incubationfor 1-2 hours at 68° C. and pelleted by centrifugation at 5000 rpm for20 minutes. The cell pellet was resuspended in 5 ml buffer A (10mMTrisHCl, pH 7.6, 1 M NaCl) at room temperature, pelleted again, andresuspended in a final volume of 2 ml buffer A. The cell suspension waswarmed to 42° C. and diluted with an equal volume of a solutioncontaining 1 % W/v low melting point agarose (Bethesda ResearchLabs,--Bethesda, Md.) in sterile water. Aliquots (100-200 μl) of thismixture were poured into molds to form agarose blocks and chilled onice. The blocks were transferred to Eppendorf tubes containing an equalvolume of lysis buffer (6 mM Tris-HCl, pH 7.6, 1 M NaCl, 100 mM EDTA, pH7.5, 0.5% w/v Bri-58 (Aldrich, Milwaukee, Wis.), 0.2% w/v sodiumdeoxycholate, 0,5% w/v sodium N-lauroylsarcosine) made from sterilestock solutions and filter sterilized following the addition ofdetergents. This solution was supplemented just prior to use with 1mg/ml lysozyme and 20 μg/ml RNase A (10 mg/ml stock in sterile dH₂ Oheated to 80° C. for 20 minutes). The cell suspension was then incubatedin the lysis buffer overnight at 37° C. The following day the lysisbuffer was removed and an equal volume of ESP buffer (0.5 M EDTA, pH9-9.5, 1% w/v in sodium lauryl sarcosinate, and 1.0 mg/ml proteinase Kpre-incubated for 2 hours at 37° C.) was added. The mixture wasincubated for 24-48 hours at 50° C. The gel block was then washed in 4changes of TE buffer (50 mM Tris-HCl, 0.1 mM EDTA, pH 7.5) containing 1mM phenyl methyl-sulfonyl fluoride (PMSF) for 4 hours at roomtemperature. The gel block was then washed twice for 4-16 hours with BamHI restriction enzyme buffer (as supplied by the manufacturer,Boehringer-Mannheim, Indianapolis, Ind.) The washed block was dissolvedin 0.5 ml of the restriction enzyme buffer at 65° C. for 10 minutes.

The restriction fragments were separated in a 2% w/v agarose gel.Southern Blot analysis included the transfer of the separatedrestriction fragments onto nitrocellulose, and hybridization with alabeled oligonucleotide probe consisting of the Bam HI restrictionfragment of the B. abortus omp2 gene locus, as shown in FIG. 3. Theresults of the Southern Blot analysis are shown in FIG. 4, and indicatethat all six species of Brucella and all B. abortus biovars tested haveconserved the omp2 locus on a 6.5 kb Bam HI fragment.

EXAMPLE 2 Heterooeneity of the omp2a Gene in Species and Biovars ofBrucella

Aliquots of Brucella DNA prepared for Example 1 were treated asdescribed in Example 1, but digested with Pst I in Pst I restrictionenzyme buffer (as provided by the manufacturer, Boeringer- Mannheim).Electrophoresis and Southern blot analysis were carried out as describedfor Example 1. The results of the Southern Blot analysis are shown inFIG. 5, and indicate that the genetic variation of the omp2 locussegregated along classical species lines, that is the Pst I restrictionfragment profiles of the omp2 gene locus were distinct for differentspecies and Biovars of Brucella. Based on Pst I restriction digestion,the species can be divided into six groups as shown in FIG. 1. Group 1includes B. abortus biovars 1,2 and 4. Group 2 includes B. abortusbiovars 3,5,6,7 and 9. Group 3 includes only B.melitensis. Group 4includes B.suis and B.neotomae. Additional restriction digestion withthe restriction enzyme Kpn I enabled distinction of Group 5, B.canisfrom the species of Group 4. Group 6 contains only B. ovis.

This data indicates that after one restriction digest with Pst I,analysis of the restriction fragments can distinguish between B.abortus,B.ovis, B.melitensis, and the remaining species of Brucella. Restrictionfragments generated from Pst1 digestion can also distinguish betweenB.abortus biovars 1 ,2 and 4 from B.abortus biovars 3,5,6, 7 and 9.Additional digestion with Kpn I permits the distinctive identificationof B.canis.

EXAMPLE 3 Detection of Brucella in Tissue Samples of Infected andControl Cattle bv Amplification of omp2 DNA

Cattle (two) (mixed breed, (Bos Taurus×Bos Indicus, Montana Beaver HeadRanch, Big Hole, Mont.), at approximately 120 days gestation, wereinfected with 1×10⁷ B.abortus S2308 organisms (obtained from Dr. BillyDeyoe, U.S.D.A. N.A.D.C.). Abomasal tissue samples were prepared fromeither aborted calves or live calves of the two infected animals and twonon-infected control animals.

Abomasal tissue samples were obtained by necroscopy following animalsacrifice. The fetal stomach or abomasum and its contents were dissectedand stored in whirlpak bags (NASCO, Fort Atkinson, Wisconsin). Portionsof the abomasal samples were heated at 68° C. for 2 hours in eppendorftubes to kill any live Brucella, and 5 μl portions were then added toamplification reactions according to the method of Saiki et al, 1985.

The standard amplification reaction was performed in a final volume of100 μl containing 200 μM of each nucleotide (dNTP, dGTP, dCTP, dTTP),2.5 units Taq polymerase, approximately 0.1ng template DNA and 1μM eacholigonucleotide primer. The reaction buffer also contained 10 mMTRIS-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, and 0.01% w/v gelatin. Theoligonucleotides used to prime the amplification were No. 32 and No. 33,as shown in FIG. 7, which amplified the omp2b gene of Brucella.

The DNA was amplified through 30 cycles including a 30 second melting at94° C., annealing over 30 seconds at 62°-65° C., and polymerization at72° C. for three minutes.

The amplified DNA was then sized in a 1-2% w/v agarose gel and stainedwith ethidium bromide to visualize the DNA. As shown in FIG. 6, DNAobtained from infected animals amplified the 1268 bp omp2b gene fragmentcharacteristic of Brucella. Non-infected animals showed no amplified DNAproduct.

EXAMPLE 4 Detection of Brucella in Milk Samples From Infected andControl Cattle

Pregnant cattle (six) (Bos Taurus×Bos Indicus, Montana Beaver HeadRanch, Big Hole, Mont.), at approximately 120 days gestation wereinfected with 1×10⁷ B.abortus S2308 (obtained from Dr. Billy Deyoe)organisms by conjunctival installation. Four uninfected cattle served ascontrols. The animals are monitored serologically for infection untilabortion or the birth of a live calf. Samples collected forbacteriologic analysis are used for PCR amplification and DNA analysisof Brucella.

Blood samples are collected weekly; sera is tested by the followingmethods:

1. buffered Brucella antigen (Card) (O'Reilly and Cunningham, 1971,Vet.Rec. 88:590-594; Ladwig, 1968, Iowa Vet. 39:9-14).

2. enzyme linked immunosorbent assay (ELIZA) (Heck et al, 1980, Am. J.Vet. Res. 41:2082-2084).

3. rivanol precipitation plate agglutination (Huber and Nicoletti, 1986,Am. J. Vet. Res, 47:1529-1531).

4. automated tube (warm) complement fixation with Brucella antigens andhemolysis in gel test (Timbs et al, 1978, N.Z. Vet. J. 26:52-56;Nicoletti and Carlsen, 1981, Am. J. Vet. Res. 42:1494-1497).

Vaginal and rectal swabs, placental and quarter milk samples from allparturient cattle will be cultured for Brucella. Rectal swabs fromviable calves, and pulmonary tissue, gastric contents, mediastinal lymphnodes, and rectal swabs from dead fetuses or neonates will be streakedonto semi-restrictive Brucella agar medium with 5% bovine serum andantibiotics (Farrell's Medium, Farrell et al, 1974, Res. Vet. Sci.16:280-286).

Culture negative parturient principals and controls will be euthanizedand at least 50 tissues will be collected, trimmed of non-lymphatictissue, and both sides of the cut surface will be rubbed over thesurfaces of Farrell's media (3 plates per tissue sample). Inoculatedmedia will be incubated at least 7 days at 37° C. in 10% CO₂ withbacterial colonies resembling Brucella further identified and biotypedby conventional methods for comparison with results for the same animalfrom PCR amplification and analysis of DNA from tissue and fluidsamples.

DNA obtained from blood, milk, semen, vaginal secretions, rectalsecretions and tissue samples will be concentrated if necessary ontonitrocellulose filters. The DNA obtained will be amplified according tothe procedure described for Example 3, using oligonucleotides whichamplify specific regions of the omp2 gene locus. Amplification of theomp2b gene locus and identification of the omp2b gene as described forExample 3 will identify the presence of Brucella organisms.Amplification of the omp2a gene using the oligonucleotides No. 34 andNo. 35, as shown in Table 2, followed by electrophoretic analysis of theamplified sequence will be used to determine the presence of Brucella inthe test sample. Restriction digestion of the amplified DNA sequenceusing the enzyme Pst I will characterize the infecting Brucella speciesas B.abortus biovars 1,2,4, B.abortus biovars 3,5,6,9. B.ovis,B.melitensis, or one of the remaining three Brucella species.Restriction digestion using the enzyme Kpn I will distinguish B.canisfrom the remaining two Brucella species, B.suis and B. neotomae.

Having described the invention above, various modifications of thetechniques, procedures, materials, and equipment will be apparent tothose in the art. It is intended that all such variations within thescope and spirit of the appended claims be embraced thereby.

We claim:
 1. A method for diagnosing Brucella infection comprising the steps of:releasing DNA from a test sample; releasing DNA from a test sample; amplifying at high stringency a gene sequence of a Brucella omp2 gene locus includes omp2a gene, omp2b gene, nucleic acid sequences intervening between the omp2a and omp2b genes, and approximately 300 nucleic acids flanking the omp2a and omp2b genes; and analyzing the amplified gene sequence to detect Brucella omp2 gene sequences diagnostic of Brucella.
 2. The method of claim 1, wherein said gene sequence corresponds to a region of Brucella omp2a gene.
 3. The method of claim 1, wherein said gene sequence corresponds to a region of Brucella omp2b gene.
 4. The method of claim 1, wherein said gene sequence is approximately 300 nucleic acids immediately adjacent to the omp2a or omp2b gene.
 5. The method of claim 1, wherein said gene sequence is the nucleic acid sequence intervening between the omp2a and omp2b genes.
 6. The method of claim 1, wherein said amplifying is by polymerase chain reaction.
 7. The method of claim 6, wherein the polymerase chain reaction is primed with an oligonucleotide pair which anneals to the omp2 gene locus of Brucella, where the omp2 gene locus includes omp2a gene, omp2b gene, nucleic acid sequences intervening between the omp2a and omp2b genes, and approximately 300 nucleic acids flanking the omp2a and omp2b genes.
 8. The method of claim 6, wherein the polymerase chain reaction is primed with an oligonucleotide pair selected from those shown in FIG.
 7. 9. The method of claim 1, wherein prior to said analyzing step, the amplified DNA is digested with a restriction enzyme to generate restriction fragments characteristic of Brucella.
 10. The method of claim 1, wherein, prior to said analyzing step the amplified DNA is digested with a restriction enzyme selected from the group consisting of Pst I and Kpn I, to generate restriction fragments characteristic of a Brucella species or biovar.
 11. The method of claim 10 wherein the restriction enzyme is Pst I.
 12. The method of claim 10 wherein the restriction enzyme is Kpn I.
 13. The method of claim 1, wherein said analyzing includes dot blot analysis using labeled DNA which hybridizes to the omp2 gene locus of rucella.
 14. The method of claim 1, wherein said analyzing includes electrophoretic separation of the amplified DNA and staining with ethidium bromide.
 15. The method of claim 1, wherein said analyzing includes Southern blot analysis using labeled DNA which hybridizes to the omp2 gene locus of Brucella.
 16. The method of claim 1, wherein said test sample is animal fluid or tissue.
 17. The method of claim 16, wherein said test sample is urine, blood, milk, semen, vaginal or rectal secretions.
 18. The method of claim 17, wherein said test sample is milk.
 19. A method for identifying a species or bovar of Brucella comprising the steps of:releasing DNA from a test sample; amplifying at high stringency a gene sequence of a Brucella omp2 gene locus from the released DNA, where the omp2 gene locus includes omp2a gene, omp2b gene, nucleic acid sequences intervening between the opm2a and omp2b genes, and approximately 300 nucleic acids flanking the omp2a and omp2b genes; and analyzing the amplified gene sequence to detect Brucella omp2 gene sequences diagnostic of a species or biovar of Brucella. 